Recently, a method of analyzing the melting temperature (Tm) of nucleic acid is used as a new method of detecting, for example, a polymorphism or a point mutation of a gene. Since this analysis method is performed through, for example, analysis of the melting curve of nucleic acid, it also is referred to as melting curve analysis or Tm analysis.
Generally, the Tm value is defined as follows. When a solution containing a double-stranded nucleic acid is heated, the absorbance at 260 nm increases. This is because heating releases the hydrogen bonds between both of the strands in the double-stranded nucleic acid to dissociate it into a single-stranded nucleic acid (i.e. to melt the nucleic acid). When all double-stranded nucleic acids are dissociated into single-stranded DNAs, the absorbance thereof is approximately 1.5 times that obtained at the start of heating (i.e. the absorbance of only double-stranded nucleic acids), which makes it possible to judge that melting is completed thereby. Based on this phenomenon, the melting temperature Tm (° C.) generally is defined as a temperature at which the absorbance has reached 50% of the total increase in absorbance.
The use of such properties of nucleic acid makes it possible to detect, for example, a polymorphism or a mutation at a target site as follows. That is, the following method is used. First, a mutant-type detection probe complementary to a target nucleic acid sequence containing a mutant-type target site is used to form a double-stranded nucleic acid between the aforementioned probe and a single-stranded nucleic acid to be analyzed. The double-stranded nucleic acid thus formed is then heat-treated, and signals indicating dissociation (melting) of the double-stranded nucleic acid accompanying the temperature rise are measured. Thereafter, the Tm value is determined from the behavior of the signal values thus measured, and from the Tm value thus determined, the presence or absence of a mutation at the target site is judged (see Nonpatent Document 1 and Patent Document 1). The higher the homology of the double-stranded nucleic acid, the higher the Tm value, and the lower the homology, the lower the Tm value. The Tm value to serve as an assessment criterion is determined beforehand with respect to each of the double-stranded nucleic acid formed of a target nucleic acid sequence containing a mutant-type target site and a mutant-type detection probe that is 100% complementary thereto and the double-stranded nucleic acid formed of a nucleic acid sequence with a wild-type target site and the mutant-type detection probe. As described above, the higher the homology, the higher the Tm value. Accordingly, the Tm value (hereinafter also referred to as the “Tmm value”) obtained in the case of the former, i.e. in the case where the target site is a mutant form, is relatively high, while the Tm value (hereinafter also referred to as the “Tmw value”) obtained in the case of the latter, i.e. in the case where the target site is a wild type, is relatively low. With respect to the double-stranded nucleic acid formed of the single-stranded nucleic acid to be analyzed and the mutant-type detection probe, signals are measured as described above and, for example, a peak in signal change is detected from a prepared melting curve. When the temperature indicating this peak, i.e. the Tm value, is comparable to the Tmm value determined beforehand, the single-stranded nucleic acid and the probe are matched 100%. That is, it can be judged that in the nucleic acid to be analyzed, the polymorphism at the target site is a mutant form. On the other hand, when the Tm value indicating the peak is lower than the Tmm value determined beforehand and is comparable to the Tmw value, the single-stranded nucleic acid and the probe are mismatched by a single base. Accordingly, it can be judged that in the nucleic acid to be analyzed, the polymorphism at the target site is a wild type. Furthermore, it also can be judged whether the polymorphism is a homozygote or a heterozygote. That is, in the case of analyzing a pair of alleles, when peaks are present around both the Tmm value and the Tmw value in the melting curve, it can be judged to be a heterozygote. On the other hand, it can be judged to be a mutant-type homozygote when the peak is present only around the Tmm value, while it can be judged to be a wild-type homozygote when the peak is present only around the Tmw value.
In this analysis, a method is employed widely in which, for example, a probe labeled with a fluorescent material is used as the aforementioned probe, and the fluorescence of the fluorescent material is measured as the signal. Generally, for such a detection method that utilizes Tm analysis, an optical detection apparatus is used that includes a detection unit for detecting the optical signal of a sample and a temperature control unit for controlling the temperature of the sample, and various such products are on the market.
As described above, in the Tm analysis, since the Tm value is determined by measuring the change in optical signal accompanying a change in the temperature, two points, i.e. whether the temperature of the sample is controlled accurately inside the optical detection apparatus and whether the optical signal of the sample is detected normally, are very important. Accordingly, with respect to the optical detection apparatus, it is indispensable to verify both the optical signal detection performance and the temperature control performance in order to maintain the reliability of the analysis results. Therefore, in the case of optical signal detection, for example, a solution containing a known fluorescent material at a known concentration is provided and the signal intensity (fluorescence intensity) of the fluorescent dye in the solution then is measured under a predetermined temperature condition, and thereby it is verified whether the optical signal can be measured normally. On the other hand, with respect to the control of sample temperature, a sample is placed in a predetermined part (hereinafter, also referred to as a “sample placement part”) of the optical detection apparatus and the temperature of the sample then is measured actually. Thus, it is verified whether the sample temperature is controlled accurately with the temperature control unit of the aforementioned apparatus.
However, such a conventional method requires that with respect to an optical detection apparatus, both the optical signal detection performance and the temperature control performance are verified separately, and therefore it takes time and energy. Furthermore, with respect to the optical signal detection performance, as described above, even when, for example, a highly reliable measured value that is comparable to the theoretical value was obtained by confirmatory measurement under a predetermined temperature condition, it does not serve as proof of temperature accuracy at the time of actual measurement. That is, it does not serve as proof that the sample placement part or the sample placed therein is actually at a predetermined preset temperature. Therefore, the measured value of the fluorescence intensity cannot be considered to indicate a highly reliable value at the “desired preset temperature”. As a result, it is indispensable to verify the temperature control performance separately. On the other hand, the temperature of the sample placed in the optical detection apparatus is measured usually with a thermometer introduced from the outside. However, there is a possibility that the temperature may vary depending on the place where the measurement is carried out in the sample placement part. Furthermore, since the sample placement part is minute, there is a possibility that the introduction itself of the thermometer from the outside may affect the actual temperature. For such reasons, it is difficult to measure the actual temperature of a sample placed in the optical detection apparatus accurately. Consequently, it is difficult to judge whether the temperature of the sample is controlled accurately by the temperature control unit.    [Nonpatent Document 1] Clinical Chemistry 46:5 631-635 (2000)    [Patent Document 1] JP 2005-58107 A